1. Principles of cloning, vectors and cloning strategies

2. DNA CLONING DNA cloning is a technique for reproducing DNA fragments.
It can be achieved by two different approaches: 
▪ cell based
  ▪ using polymerase chain reaction (PCR). 
a vector is required to carry the DNA fragment of interest into the host cell.    

3. DNA CLONING
DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount.
This technique is the first stage of most of the genetic engineering experiments:
▪ production of DNA libraries
▪ PCR
▪ DNA sequencing 

4. DNA CLONING Massive amplification of DNA sequences
Stable propagation of DNA sequences
A single DNA molecule can be amplified allowing it to be:
▪ Studied - Sequenced
▪ Manipulated - Mutagenised or Engineered
▪ Expressed - Generation of Protein

5. CLONING PROCESS Gene of interest is cut out with RE
Host plasmid is cut with same RE
Gene is inserted into plasmid and ligated with ligase
New plasmid inserted into bacterium (transform)

6. PLASMID CLONING STRATEGY
Involves five steps: Enzyme restriction digest of DNA sample. Enzyme restriction digest of DNA plasmid vector. Ligation of DNA sample products and plasmid vector. Transformation with the ligation products. Growth on agar plates with selection for antibiotic resistance.

7. STEP 1. RE DIGESTION OF DNA SAMPLE

8. STEP 2. RE DIGESTION OF PLASMID DNA

9. STEP 3. LIGATION OF DNA SAMPLE AND PLASMID DNA

10. STEP 4. TRANSFORMATION OF LIGATION PRODUCTS
The process of transferring exogenous DNA into cells is call “transformation”
There are basically two general methods for transforming bacteria. The first is a chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells.
A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake.

11. CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

12. TRANSFORMATION BY ELECTROPORATION

13. STEP 5. GROWTH ON AGAR PLATES

14. STEP 5
Blue colonies represent Ampicillin-resistant bacteria that contain pVector and express a functional alpha fragment from an intact LacZ alpha coding sequence. White colonies represent Ampicillin-resistant bacteria that contain pInsert and do not produce LacZ alpha fragment

15. TERMS USED IN CLONING DNA recombination.
DNA recombination. 
The DNA fragment to be cloned is inserted into a vector. 
Transformation. 
The recombinant DNA enters into the host cell and proliferates.
Selective amplification. 
A specific antibiotic is added to kill E. coli without any protection.  The transformed E. coli is protected by the antibiotic-resistance gene
Isolation of desired DNA clones  

16. CLONING VECTORS
Cloning vectors are DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube.
Cloning vectors share four common properties:
1. Ability to promote autonomous replication. 2. Contain a genetic marker (usually dominant) for selection. 3. Unique restriction sites to facilitate cloning of insert DNA. 4. Minimum amount of nonessential DNA to optimize cloning.

17. PLASMIDS
Bacterial cells may contain extra-chromosomal DNA called plasmids.
Plasmids are usually represented by small, circular DNA.
Some plasmids are present in multiple copies in the cell

18. PLASMID VECTORS Plasmid vectors are ≈1.2–3kb and contain:
replication origin (ORI) sequence
a gene that permits selection,
Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin.
Exogenous DNA can be inserted into the bracketed region .

19. SELECTIVE MARKER
Selective marker is required for maintenance of plasmid in the cell.
Because of the presence of the selective marker the plasmid becomes useful for the cell.
Under the selective conditions, only cells that contain plasmids with selectable marker can survive
Genes that confer resistance to various antibiotics are used.
Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

20. ORIGIN OF REPLICATION
Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes.
Without replication origin, DNA cannot be replicated in the cell.

21. MULTIPLE CLONING SITE
Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other
Restriction sites of the polylinker are not present anywhere else in the plasmid.
Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

22. MULTIPLE CLONING SITE
Gene to be cloned can be introduced into the cloning vector at one of the restriction sites present in the polylinker

24. TYPES OF CLONING VECTORS

25. CLONING VECTORS
Different types of cloning vectors are used for different types of cloning experiments.
The vector is chosen according to the size and type of DNA to be cloned

26. PLASMID VECTORS
Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100bp -10kb).
ColE1 based, pUC vehicles commercially available ones, eg pGEM3, pBlueScript

27. Disadvantages using plasmids
Cannot accept large fragments
Sizes range from kb
Standard methods of transformation are inefficient

28. BACTERIOPHAGE LAMBDA
Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that uses E. coli as host.
Its structure is that of a typical phage: head, tail, tail fibres.
Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA "sticky end" at both ends; these ends are complementary in sequence and can hybridize to each other (this is the cos site: cohesive ends).
Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected.
The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host.

29. BACTERIOPHAGE LAMBDA

30. COSMID VECTOR Purpose: 1. Clone large inserts of DNA: size ~ 45 kb
Features: Cosmids are Plasmids with one or two Lambda Cos sites.
Presence of the Cos site permits in vitro packaging of cosmid DNA into Lambda particles

31. COSMID VECTOR Thus, have some advantages of Lambda as Cloning Vehicle:
Strong selection for cloning of large inserts
Infection process rather than transformation for entry of chimeric DNA into E. coli host
Maintain Cosmids as phage particles in solution
But Cosmids are Plasmids: Thus do NOT form plaques but rather cloning proceeds via E. coli colony formation

32. Yeast Artificial Chromosomes

33. Yeast Artificial Chromosomes
Purpose:
Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes
Clone very large inserts of DNA: 100 kb - 10 Mb
Features: YAC cloning vehicles are plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome

34. Additional features:
Often have a selection for an insert
YAC cloning vehicles often have a bacterial origin of DNA replication (ori) and a selection marker for propogation of the YAC through bacteria.
The YAC can use both yeast and bacteria as a host

35. PACs and BACs PACs - P1-derived Artificial Chromosomes
E. coli bacteriophage P1 is similar to phage lambda in that it can exist in E. coli in a prophage state.
Exists in the E. coli cell as a plasmid, NOT integrated into the E. coli chromosome.
P1 cloning vehicles have been constructed that permit cloning of large DNA fragments- few hundred kb of DNA
Cloning and propogation of the chimeric DNA as a P1 plasmid inside E. coli cells
BACs - Bacterial Artificial Chromosomes
These chimeric DNA molecules use a naturally-occurring low-copy number bacterial plasmid origin of replication, such as that of F-plasmid in E. coli.
Can be cloned as a plasmid in a bacterial host, and its natural stability generally permits cloning of large pieces of insert DNA, i.e. up to a few hundred kb of DNA.

36. RETROVIRAL VECTORS
Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells.
Retroviruses are RNA viruses.
The viral RNA is converted into DNA by the viral reverse transcriptase and then is efficiently integrated into the host genome
Any foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there practically indefinitely.
Retroviral vectors are widely used to study oncogenes and other human genes.

37. Types of expression systems
Bacterial:  plasmids, phages
Yeast:  expression vectors:  plasmids, yeast artifical chromosomes (YACs)
Insect cells:  baculovirus, plasmids
Mammalian:
viral expression vectors (gene therapy):
SV40
vaccinia virus
adenovirus
retrovirus
Stable cell lines (CHO, HEK293)

38. EXPRESSION VECTORS
Allows a cloned segment of DNA to be translated into protein inside a bacterial or eukaryotic cell.
Vectors will contain the ff:
(a) in vivo promoter
(b) Ampicillin selection
(c) Sequencing primers

39. EXPRESSION VECTORS Produces large amounts of a specific protein
Permits studies of the structure and function of proteins
Can be useful when proteins are rare cellular components or difficult to isolate

40. Common problems with bacterial expression systems
Low expression levels:
▪ change promoter
▪ change plasmid
▪ change cell type
▪ add rare tRNAs for rare codons on second plasmid
Severe protein degradation:
use proteasome inhibitors and other protease inhibitors
try induction at lower temperature
Missing post-translational modification:  co-express with kinases etc.
Glycosylation will not be carried out:
use yeast or mammalian expression system
Misfolded protein (inclusion bodies):
co-express with GroEL, a chaperone
try refolding buffers

41. REPORTER GENE VECTORS
A gene that encodes a protein whose activity can be easily assayed in a cell in which it is not normally expressed
These genes are linked to regulatory sequences whose function is being tested
Changes in transcriptional activity from the regulatory sequences are detected by changes in the level of reporter gene expression

42. SHUTTLE VECTORS
Shuttle vectors can replicate in two different organisms, e.g. bacteria and yeast, or mammalian cells and bacteria.
They have the appropriate origins of replication.
Hence one can clone a gene in bacteria, maybe modify it or mutate it in bacteria, and test its function by introducing it into yeast or animal cells.

43. CLONING STRATEGY
Strategy depends on the starting information and desired endpoint.
Starting Information or Resources:
▪ Protein sequence
▪ Positional cloning information
▪ mRNA species / sequence
▪ cDNA libraries
▪ DNA sequence known or unknown
▪ genomic DNA libraries
▪ PCR product

44. How Are Genes Cloned Using Plasmids?
To understand how genes are cloned, we need introduce three terms.
Recombinant DNA- is mixed DNA
Vector -it carries recombinant DNA into cells.
Plasmids - are tiny circular pieces of DNA that are commonly found in bacteria.

45. Why Plasmids are Good Cloning Vectors
small size (easy to manipulate and isolate)
circular (more stable)
replication independent of host cell
several copies may be present (facilitates replication)
frequently have antibody resistance (detection easy)

46. How is foreign DNA Inserted into a Plasmid?
To open up the DNA a restriction enzyme is used.
Cut the DNA at a specific place called a restriction site.
The result is a set of double-stranded DNA pieces with single-stranded ends
These ends that jut out are not only "sticky" but they have gaps that can be now be filled with a piece of foreign DNA
For DNA from an outside source to bond with an original fragment, one more enzyme is needed
DNA ligase seals any breaks in the DNA molecule

47. RESTRICTION ENZYMES
Restriction enzymes: enzymes that cut DNA in specific places function:
Inactivate foreign DNA
Breaks only palindrome sequences, i.e. those exhibiting two-fold symmetry
Important in DNA research, i.e. sequencing, hybridization
Companies purify and market restriction enzymes

48. RESTRICTION ENZYMES

49. CLONING METHODOLOGY
Cut the cloning vector with R.E. of choice, eg Eco RI
Cut DNA of interest with same R.E. or R.E. yielding same sticky ends, e.g. Bam HI and Sau 3A
Mix the restricted cloning vector and DNA of interest together.
Ligate fragments together using DNA ligase
Insert ligated DNA into host of choice - transformation of E. coli
Grow host cells under restrictive conditions, grow on plates containing an antibiotic

54. BLUE/WHITE SCREENING
Colony Selection: finding the rare bacterium with recombinant DNA
Only E. coli cells with resistant plasmids grow on antibiotic medium
Only plasmids with functional lacZ gene can grow on Xgal  lacZ(+) => blue colonies lacZ functional => polylinker intact => nothing inserted, no clone lacZ(-) => white colonies polylinker disrupted => successful insertion & recombination!

55. α -complementation
The portion of the lacZ gene encoding the first 146 amino acids (the α -fragment) are on the plasmid
The remainder of the lacZ gene is found on the chromosome of the host.
If the α -fragment of the lacZ gene on the plasmid is intact (that is, you have a non-recombinant plasmid), these two fragments of the lacZ gene (one on the plasmid and the other on the chromosome) complement each other and will produce a functional β -galactosidase enzyme.

56. SCREENING RECOMBINANTS
In the example shown above, the b-galactosidase gene is inactivated. The substrate "X-gal" turns blue if the gene is intact, ie. makes active enzyme. White colonies in X-gal imply the presence of recombinant DNA in the plasmid.

57. COMPLICATIONS lacZ gene not expressed constitutively
X-gal does not activate gene expression
must use IPTG as inducer
(isopropyl-β-D-thio-galactoside)
small inframe insertions may not inactivate α peptide
still get blue colonies (often lighter – less activity

58. ENZYMES USED IN MOLECULAR BIOLOGY
Alkaline phosphatase
Removes phosphate groups from 5' ends of DNA (prevents unwanted re-ligation of cut DNA)
DNA ligase
Joins compatible ends of DNA fragments (blunt/blunt or complementary cohesive ends). Uses ATP
DNA polymerase I
Synthesises DNA complementary to a DNA template in the 5'-to-3'direction. Starts from an oligonucleotide primer with a 3' OH end
Exonuclease III
Digests nucleotides progressiviely from a DNA strand in the 3' -to-5' direction
Polynucleotide kinase
Adds a phosphate group to the 5' end of double- or single-stranded DNA or RNA. Uses ATP
RNase A
Nuclease which digests RNA, not DNA
Taq DNA polymerase
Heat-stable DNA polymerase isolated from a thermostable microbe (Thermus aquaticus)

59. ENZYMES USED IN MOLECULAR BIOLOGY

60. RESTRICTION ENZYMES
The restriction enzymes most used in molecular biology labs cut within their recognition sites and generate one of three different types of ends.

61. 5’ OVERHANGS
5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends. Bam HI cuts in this manner.

62. 3’ OVERHANGS
3' overhangs: Again, we see asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends. KpnI cuts in this manner.

63. BLUNT ENDS
Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs. SmaI is an example of an enzyme that generates blunt ends.

64. Converting a 5’ overhang to blunt end
Both Klenow and T4 DNA polymerase can be used to fill in 5’ protruding ends with dNTPs
Used in joining DNA fragments with incompatible ends
Once the ends have been blunted, ligation can proceed

65. Converting a 3’ overhang to a blunt end
T4 DNA polymerase has a 3’-5’ exonuclease activity
In the presence of excess dNTPs will convert a 3’ protruding end to a blunt end
Ligation can know proceed

66. DIRECTIONAL CLONING
Often one desires to insert foreign DNA in a particular orientation
This can be done by making two cleavages with two different restriction enzymes
Construct foreign DNA with same two restriction enzymes
Foreign DNA can only be inserted in one direction

67. Good efficiency of ligation of foreign DNA into a vector can be achieved if both the vector and the insert DNA are cut with 2 different restriction enzymes which leave single stranded ends (cohesive ends).
The DNA is ligated in only one direction, and there is only a low background of non-recombinant plasmids.
If only one restriction enzyme is used to cut the vector and insert, then efficiency of ligation is lower, DNA can be inserted in two directions and tandem copies of inserts may be found.
To avoid high background of non-recombinants, alkaline phosphatase is used to remove 5' phosphate groups from the cut vector to prevent self-ligation.

68. Alkaline phosphatase
Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH

69. Alkaline phosphatase
There are two primary uses for alkaline phosphatase in DNA manipulations:
Removing 5' phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme. In subsequent ligation reactions, this treatment prevents self-ligation of the vector and thereby greatly facilitates ligation of other DNA fragments into the vector (e.g. subcloning).
Removing 5' phosphates from fragments of DNA prior to labeling with radioactive phosphate. Polynucleotide kinase is much more effective in phosphorylating DNA if the 5' phosphate has previously been removed

70. DEPHOSPORYLATED VECTOR

71. R.E.S WITH COMPATIBLE ENDS

72. Generating a new R.E. site at a blunt end
Use linkers to generate a new R.E.
Linkers are used to place sticky ends on to a blunt-ended molecule
Short blunt ended synthetic ds DNA containing a R.E. site
Experimental design:
(i) Blunt ended DNA + linker (T4 ligase)
(ii) Digest with appropriate R.E.
(iii) Ligate to vector

74. Introducing a R.E. site by PCR
R.E. site is designed into the 5’ end of the PCR primer
PCR fragment is digested with appropriate R.E., purified and ligated into plasmid vector

75. USING DIFFERENT R.E.s

76. It depends on the enzyme
Some catalogues of enzymes provide anecdotal data on the efficiency of enzymes trying to work at the ends of DNA molecules.
Generally, enzymes work better if they have a couple of extra nucleotides at the end - they don't do very well if they are perched on the end of a molecule.

77. Ligation of foreign DNA to plasmid vectors
Termini on foreign DNA
Requirements for cloning
Comments
Blunt -ended
High conc of DNAs and ligase
Large no. of non-recombinant clone
R.E. sites may be eliminated
Tandem copies of foreign DNA
Different protruding termini
Requires purification of plasmid after digestion
R.E. sites at junctions are conserved
Low no. of non-recombinants
Foreign DNA is inserted in only one orientation
Identical protruding termini
Phosphatase treatment of linear plasmid vector
Foreign DNA is inserted in either orientation

78. IDENTIFICATION OF POSITIVE CLONES
One of the first steps is to identify clones carrying the recombinant plasmid, with the desired DNA insert.
This can be done by 'picking' clones - choosing individual bacterial colonies in order to isolate the plasmid DNA from each of them.
Single bacterial colonies are grown in culture broth containing the selection antibiotic in order to maintain the plasmid.
The plasmid DNA is extracted by the standard minipreparation technique and then analysed by restriction digest.
After digesting the DNA, different sized fragments are separated by agarose gel electrophoresis and the sizes determined by comparison with known DNA molecular weight marke

79. AGAROSE GEL ELECTROPHORESIS

81. RECOMBINANT DNA R.E. are a useful tool for analysing Recombinant DNA
▪ checking the size of the insert
▪ checking the orientation of the insert
▪ determining pattern of restriction sites within insert
Sometimes it is important to determine the orientation of the DNA insert in relation to the vector sequence.
This can be done simply by restriction digest using enzyme(s) which cut the vector sequence near to the insert and cut within the insert sequence (asymmetrically).

82. APPLICATIONS Cloning DNA fragments
Generating Libraries: essential step for genome mapping
Positional cloning – discovering disease genes
Discovering genes from e.g. Protein sequence

83. PCR cloning strategies
Cloning methods for PCR products are divided into three types:
(i) blunt-end cloning
(ii) sticky-end cloning
(iii) T-A cloning

84. PCR Cloning Considerations
Nature of the Insert: not all PCR fragments will clone with the same efficiency into the same vector.
Insert Size:The size of the fragment being cloned is a primary contributor to the overall cloning efficiency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a much lower efficiency.
Vector-to-Insert Ratio:Optimization of molar concentration ratios of the vector to insert is critical to ensure efficient cloning. insert ratios: 1:1, 1:3,

85. T-A Cloning
When DNA fragments are generated Taq polymerase adds 1 or 2 extra adenines onto the end of 3’ end of blunt ds DNA
Several commercially available kits take advantage of this ability
Use a plasmid vector with thymidine residues linked onto the 3’ ends of linearised plasmid DNA

86. TA CLONING VECTOR

87. TA RECOMBINANT VECTOR

88. ANALYSIS OF CLONED DNA Is it the one you wanted?
 What are its molecular characteristics?

89. Restriction mapping: determining the order of restriction sites in a cloned fragment:
Gel electrophoresis: separates DNA fragments by molecular weight
Southern Blot analysis: DNA is transferred ("blotted") to filter paper.Filter is exposed to a DNA probe. Binds specifically to target DNA immobilized on filter
DNA sequencing: provides complete order of bases in a DNA fragment       

91. DNA FORMS OF A PLASMID Uncut plasmid DNA can be in any of five forms:
▪ nicked
▪ circular
▪ linear covalently closed
▪ supercoiled
▪ circular single-stranded.
The exact distances between the bands of these different forms is influenced by:
▪ percentage of agarose
▪ time of electrophoresis
▪ degree of supercoiling
▪ the size of the DNA.
Linear band = linear size of plasmid

92. CLONING INTO A PLASMID